ABSTRACT
Fluorescence microscopy is an invaluable and extremely popular tool in biological sciences due to its ability to noninvasively image the interior of cells in all spatial dimensions. Attaching uorophores to the molecules of interest with antibody labeling or by genetic modi cation is highly speci c, enabling sensitive me asurements w ith low bac kground. Until re cent ye ars, however, t he re solution of uorescence microscopy has been conceptually a nd practically l imited by t he wave nature of l ight to a bout half the wavelength of the light used to form the image. Ernst Abbe [1], Émile Verdet, and Lord Rayleigh described this di raction l imit quantitatively, g iving the lateral extent of the l ight distribution in the focus, ∆r, as ∆r ≈ λ/(2nsin α), where λ is the wavelength of light used, n the index of refraction of the surrounding medium, and α the half-angle subtended by the outermost rays focused by the lens. us, the details of structures smaller than this dimension could not b e seen by the far- eld optical microscope, preventing the observation of interactions between cellular constituents that take place below the di raction limit.
